A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells

Okayama et al. (1983). Mol. Cell. Biol. DOI: 10.1128/mcb.3.2.280-289.1983 Citations: 50

cDNA cloning vectors for E. coli lacked mammalian expression capability. The pcD vector combined an SV40 promoter, splice sites, and a poly(A) signal for both. A pcD-alpha-globin construct gave spliced mRNA in COS cells, and a pcD library yielded a functional HPRT clone.

Parts used

SV40 Late Intron

Key findings

  • The pcD vector combined the SV40 early promoter with two late region introns (16S and 19S splicing sequences) positioned upstream of a cDNA cloning site, plus an SV40 late polyadenylation signal downstream. This arrangement enabled cDNA inserts to undergo transcription initiation, intron splicing, and 3' polyadenylation in mammalian cells while retaining compatibility with E. coli cloning via pBR322 origin and ampicillin resistance marker.

  • Transfection of pcD-α-globin into COS cells produced α-globin transcripts that initiated correctly at the SV40 promoter, spliced at either of the two introns, and polyadenylated either at the globin-encoded signal or the distal SV40 polyadenylation site. RNA mapping confirmed proper transcript processing for the inserted cDNA, demonstrating the vector's ability to generate fully processed mRNAs from cDNA sequences lacking native regulatory elements.

  • A cDNA library constructed with mRNA from SV40-transformed human fibroblasts yielded approximately 1.4 × 10^6 clones, including full-length cDNA sequences encoding functional proteins. The library successfully identified and expressed hypoxanthine-guanine phosphoribosyltransferase (HPRT), validating the approach for isolating rare cDNAs based on protein function rather than hybridization screening.

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Abstract

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).